TF (typhoid fever) in man is the clinical manifestation of a generalized or systemic infection by Salmonella typhi, a gram-negative bacterium which penetrates the organism through the gastrointestinal tract, usually by ingestion of water or food contaminated by human feces. S. typhi belongs to the serotype 9, 12, d, Vi, defined by the repeated sugar units (9, 12) of the O antigen, that together with lipid A constititutes the lipopolysaccharide (LPS) of the outer membrane; by the H antigen (d) constituted by the flagellar protein or flagellin, and by the Vi antigen or K capsular polysaccharide (Calva, E. et al., 1988, Research opportunities in typhoid fever: Epidemiology and Molecular Biology. BioEssays 9: 173-177).
As other gram-negative bacteria, S. typhi has three envelopes, constituted by two membranes, the internal and external, and an intermediate cell wall or peptidoglycan.
One of the major S. typhi outer membrane proteins (mOMPs) is OmpC (Puente J.L. et al., 1987, Isolation of an OmpC-like outer membrane protein gene from Salmonella typhi. Gene 61: 75-83.) The composition of the gene that codifies OmpC is very similar to that from E. coli (Puente, J.L. et al.) Comparative analysis of the Salmonella typhi and Escherichia coli OmpC genes. Gene 83: 197-206). OmpC (a porin) in E. coli forms a trimer that constitutes a 1.1 amstrong-diameter pore, which allows the passing of hydrophilic molecules. In E. coli, OmpF (a porin) forms trimers that constitute 1.2 amstrong-diameter pores. Another mOMP is OmpA which is a structural monomer.
In addition, in both: E. coli and in S. typhimurium exist a variety of proteins, some of which are regulated by metabolites such as calcium, phosphate, iron, maltose and others. To this respect, in the case of iron (Fe) it has been observed that there is a competition for this metal, between the host and the invader in such a way that both have developed different mechanisms for its acquisition or its sequestering during infection (Bullen, J.J., 1981, The significance of iron in infection. Rev. Infect. Dis. 3: 1127-1138; Weinberg, E.D., 1978, Iron and Infection. Microbiol. Rev. 42: 45-66).
It is evident that typhoid fever affects individuals from different geographical areas, ages and socioeconomical levels; thus there is in consequence a great need for new, highly sensitive and specific, rapid, and easy to perform diagnostic tests, for detecting TF in such a manner that it can be easily distinguished from other febrile diseases. This is even more important for children, in view that they tend to develop mild forms of the disease (Ferreccio, C. et al., 1984, Benign bacteremia caused by S. typhi and S. paratyphi in children younger than two years. J. Pediatr. 104: 899-901). Due to the fact that the majority of the population in areas where TF is endemic has high levels of serum antibodies against S. typhi, induced by its continuous exposure to the microorganism, the serological tests performed in these areas are of low specificity for the diagnosis of TF. Moreover, a significant increase in the antibody titers against the O antigen usually is detected until the second or third week after onset of fever. (Calva et. al. 1988 Research Opportunities in typhoid Fever: Epidemiology and Molecular Biology. Bioessays 9:173-177).
To date, the most exact diagnostic test for TF is the isolation of S. typhi from bone marrow aspirates, which has a 70 to 90% sensitivity and specificity. Nevertheless, it is an aggressive procedure and can only be performed in some hospitals, thus it is an impractical test. Blood cultures or hemocultures are more commonly used and easy to perform, although their sensitivity is also 70-90% when three consecutive cultures are done, at 1-2 day intervals. The important disadvantages related to this method are that the isolation and identification of S. typhi takes at least 72 hours and that the hemocultures might not be highly sensitive, due to a low concentration of circulating S. typhi in blood (approximately 20 cells/ml or less), especially when the patients have taken antibiotics before the culture, a common situation in many countries (Edelman, R. and Levine M.M., 1986, Summary of an international workshop on typhoid fever. Rev. Infect. Dis. 8: 329-350).
In some investigations performed with different antigenic reagents, of non-proteic nature, and with different methodologies, varied results have been observed. For instance, one of the most used serodiagnostic methods for TF, and one of the oldest, is the Widal test or "febrils reactions", that consists in the detection of agglutination in a suspect serum with the O and H antigens. With this test it is possible to diagnose enteric fever mainly by S. typhi and S. paratyphi. Nevertheless, due to the elevated background titers in healthy individuals in endemic areas, its use is recommended for individuals from non-endemic areas and to persons below ten years of age in endemic areas (Levine M.M. et al., 1978, Diagnostic value of the Widal test in areas endemic for typhoid fever. Am. J. Trop. Med. Hyg. 27: 795-800).
The counter immuno electrophoresis (CIE) method has also been used, utilizing various antigenic extracts. With an antigenic extract obtained by sonication, the best results were obtained, i.e. a sensitivity and specificity for TF of 70 and 96%, respectively (Talwar, V. et al, 1986, Counter ion immuno electrophoresis (CIEP) for serological diagnosis of typhoid fever. Indian J. Med. Res. 84: 353-357). By solid-phase radioimmuno assay (RIA), positive values were obtained among 94% and none of TF patients and healthy controls, respectively. In contrast, the same values for the Widal test were 81 and 25% (Tsang, R.S.W. et al., 1981, Antibody response to the lipopolysoccharide and protein antigens of S. typhi during typhoid infection. Clin. Exp. Immunol. 46: 508-514). Another group found that CIEP had a sensitivity of 90% for diagnosing TF in culture-negative clinically diagnosed TF patients, as compared with 48% obtained with the Widal test (Srivastava, V.K. et al., 1986, Comparison of counter current immunoelectrophoresis and Widal tests in the diagnosis of typhoid fever in childhood. Indian J. Pathol. Microbiol. 29: 21-26).
The ELISA (enzyme-linked immunosorbent assay) has been used by different groups interested in the diagnosis of TF, using practically all the surface antigens described, treated or obtained by variable ways, has led to the obtention of variable results.
Beasley, W.J. et al. (1981, Improved serodiagnosis of Salmonella enteric fevers by an enzyme-linked immunosorbent assay. J. Clin. Microbiol. 13: 106-114), performed an ELISA using a proteic antigen. In their work they developed tests with TF and paratyphoid (PTF) patients; they could detect as positives some samples that appeared to be false negatives by the Widal (agglutination) test. Nevertheless, the percent of positive values was indistinct for TF and for PTF and, on the other hand, there was great dispersion among the positive values; for this reason it was not possible to propose a cutoff line at one serum dilution. Also, when the immune response was evaluated by immunoglobulin G (IgG) and by immunoglobulin M (IgM), no significant difference was observed in the IgM and IgG titers between sera from acute and convalescent phase individuals. Lastly, sera from persons with other kinds of infections different from enteric fever were not evaluated.
Calderon, I. et al. (1986, Antibodies to porin antigens of S. typhi induced during typhoid fever in humans. Infect. Immun. 52: 209-212), titrated the immune response to S. typhi OMPs with IgG and IgM by ELISA, and found that the absorbance values obtained with porins, presumably free of lipopolysaccharide (LPS), with sera positive for TF, differed significantly from control sera of clinically healthy individuals from an endemic area. They also compared this response with that obtained against the LPS and flagellin, observing a greater response against the porins. Nevertheless, in their assay they did not evaluate subjects with other kinds of infections.
Appassakij, H. et al. (1987, Enzyme-linked immunosorbent assay for detection of S. typhi protein antigen. J. Clin. Microbiol. 25: 273-277), designed an ELISA method for the determination of proteic antigen in serum. When they tried it on groups of subjects with TF, PTF, other febrile diseases, as well as in healthy controls, they observed a great dispersion in the TF and PTF groups and a certain degree of crossing-over when a cutoff value was established. They obtained an 84% sensitivity and an 89% specificity.
The ELISA was tested by Banchuin, N. S. et al. (1987Detection of S. typhi protein antigen in serum and urine: a value for diagnosis of typhoid fever in an endemic area. Asian Pacific J. Allergy Immunol. 5: 155-159), for detecting antigen in serum and in urine; and they compared it with the Widal test. With this assay they obtained a predictive positive value of 33% in serum and 64% in urine; against 17% in Widal-O and 13% in Widal-H. Their negative predictive value was 97% in serum and urine, and of 97% in the Widal reactions. With these results, they demonstrated that the assay was significantly superior to the Widal test in the positive predictive value, and that the Widal is of low value for adults in endemic areas, as previously pointed out by Levine, M.M. et al. (1978, Diagnostic value of the Widal test in areas endemic for typhoid fever. Am. J. Trop. Med. Hyg. 27: 795-800), and Lambertucci, J.R. et al. (1985, The value of the Widal test in the diagnosis of prolonged septicemic salmonellosis. Rev. Inst. Med. Trop. Sao Paulo 27: 82-85).
Use of the ELISA for detecting antibodies to Salmonella typhi lipopolysaccharide (LPS) has been reported. In two reports the LPS-ELISA was found to be more specific and more sensitive, respectively than the Widal test. In one study, the % of serum samples positive for LPS immunoglobulins ranged between 83 and 97% versus 0 to 4% in healthy controls; for the Widal test these values ranged between 41 and 90%, and 0 and 4%, respectively, although they were obtained at lower dilution of the test serum. Nevertheless, there was wide scattering of the data, making it difficult to set a cutoff value between positive and negative values. In addition, lower dilutions of the test serum, than the ones reported below in the FT-ELISA described in this invention, were used for the LSP-ELISA (Nardiello, S. et al., 1984, Serodiagnosis of typhoid fever by enzyme-linked immunosorbent assay determination of anti-Salmonella typhi lipopolysaccharide antibodies. J. Clin. Microbiol. 20: 718-721). In another report, even lower dilutions of the test serum were used, wide scattering of the data was obtained, and positive values for only 73 to 82% of the bacteriologically proven cases were obtained. Nevertheless, positive values with the Widal test were present in only 41% of the samples (Srivastava, L. and Srivastava, V.K., 1986, Serological diagnosis of typhoid fever by enzyme-linked immunosorbent assay [ELISA]. Annals of Tropical Paediatrics 6: 191-194).
After analyzing the above mentioned data with respect to the protein-ELISAs, one can conclude that, in spite of the various investigations in this field, performed mainly in areas where TF is endemic, and of the important efforts that have been made for diagnosing efficiently this disease, there is still no diagnostic system for TF that is rapid, sensitive, specific, reproducible, practical, and economical. Thus, a process has been developed for obtaining and utilizing an antigenic reagent that, due to its characteristics, allows the indirect determination of Salmonella typhi, the casual agent of TF.